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Home , Action potential, Bernard Katz

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On Significant Discoveries and Landmark Experiments in the Development of Modern

John DiCecco Department of Electrical and Computer Engineering, University of Rhode Island, Kingston, RI 02881-0805 USA

Hermann von Helmoltz, and Ernst von Brücke, would prove Abstract- The methodologies utilized in the contemporary to be the significant “growth spurt” of neuroscience [2]. Their study of neuroscience are manifest from the collective efforts collective efforts to relate biological processes to the fields of of centuries of determined and insightful researchers. From chemistry and physics created a new understanding of the the first query into the most basic of all mysteries, what is a neurophysiologic process. Many of their insights and soul, scientists have struggled to narrow the search in what is discoveries are still valid, proving the true genius of their an ever expansive library of possibilities. The early works of work. Galen, Descartes, and Newton helped focus that attention The roots of the landmark achievements of Bernstein, toward the and the elaborate network of Curtis, Cole, Hodgkin, Huxley, and Katz can all be linked to interconnections the brain uses to interface with the outside the tour de force of 19th century biophysics. In turn, the world. Later works of Reymond, Hermann, Helmholtz, contemporary understanding of neuroscience can be attributed Hodgkin and Huxley, and many others, helped form the framework of the current understanding of neuroscience. This to these pioneers, whose work is still considered valid and paper will attempt to explore the contributions of the critical applicable to the currently accepted models of neurobiology. physiological experiments that facilitated the advancement of It is therefore postulated that only marginal advancement in this understanding. It is the further goal of this paper to the true understanding of neurological processes can take establish, by citation of the discoveries throughout the history place without a fair treatment of the history of the science. of neuroscience, that the contemporary understanding of the activation, creation, and conduction of action potentials II. IN THE BEGINNING through neural tissue is valid. The first plenary work on the brain and the suggestion of its function was offered by Galen in 177 C.E. His work with Index Terms— History, neuroscience, , the Barbary ape led to an understanding of many of the , , . physiological processes of the human anatomy, including the brain. I. INTRODUCTION The investigation into the action of the brain would have to wait until the 1600s, when a young philosopher and N 159 C.E. a young physician was appointed the personal mathematician named Rene Descartes suggested that the brain I (actually the pineal gland) was responsible for controlling the doctor to Marcus Aurelius, emperor of Rome. He was to care body and mind. He described the body as a machine and the not only for the Emperor but for the gladiators who fought for acted as force transducers. Researchers and scientists, his amusement. Over many years he made tremendous such as Leeuwenhoek and Robert Hooke, discoverer of the contributions to the understanding of human by , armed with microscopes and a penchant for discovery, set working mostly with the Barbary ape, and applying those out to identify the mechanisms of this control. discoveries to humans [1]. He would discover that the Among the many contributions Isaac Newton has made to kidneys produced urine by tying the ureter closed and the scientific process, his theory on conduction is the observing the kidney swell. He would go on to discover that most important to the subject of neuroscience. He believed, as of anatomical structures would result after severing did Borelli and Leeuwenhoek, that nerves were solid and the nerves that controlled them, though he did not fully continuous and his thoughts on electrical pulses propagating understand their functionality. These discoveries seem so through nerves had a profound impact on , often st trivial in the 21 century but to Galen, the young doctor, they considered the father of neuroscience. Galvani had used a were revolutionary. Still many others have contributed in this Leyden jar, a predecessor to the modern capacitor, to store manner and the contemporary model of biological processes is from a lightning storm. He then wired the jar to a indebted to their discoveries. severed ’s limb in a dramatic display of the ability of As with all scientific progress, the advancement of nerves to conduct electricity (figure 1). Though this understanding is not a linear one. The biophysics movement experiment is not the first of its kind, it is generally considered of 1847, led by Carl Ludwig, Émil du Bois-Reymond,

2 to be the first well documented and well executed. however, had been approaching the issue of electrochemical properties from the ionic perspective, hypothesizing that osmotic properties were a result of molecular pores. It would be the work of Julius Bernstein, a student of du Bois- Reymond and assistant to Helmholtz, which would help unify the results of Helmholtz, Reymond and Brücke and define the modern understanding of electrochemical transmission. Bernstein would first need to incorporate the seminal work of Walther Nernst.

IV. DIFFUSION, GRADIENTS, AND THE MEMBRANE POTENTIAL Figure 1. Galvani’s frog.

The latter part of the 17th century into the early 18th century The work of Wilhelm Ostwald and his artificial semi- spawned a craze for electrical experiments, both for the permeable membrane established the notion of selective relationship to the physiological implications as well as the permeability and that the electrical potential seen at these nature of electricity itself. Ewald Von Kleist and Pieter van membranes was a result of the permeability of [4]. Musschenbroek devoted much of their time to the During this time, Walther Nernst, a noted German physicist and chemist, developed the ideas that would eventually lead development of the Leyden jar. With the ability to store him to an electrochemical potential formula, known as the electrical charge, scientists like , Charles . He proposed that the equilibrium potential Augustin de Coulomb, Andre Marie Ampere, and Georg across a semi-permeable membrane varies in a linear relation Simon Ohm, set out to describe, in great detail, the ship with the absolute temperature and in a logarithmic mechanisms for the movement of charge [3]. In the midst of relationship with respect to the ionic concentration on either this research, two schools of thought emerged; that of an side of the membrane. In algebraic form electricity and that of a two metal electricity. After Volta succeeded in making the first dc battery from discs of copper and zinc, the idea of animal electricity was suppressed. RT [ ]outside Eion = ln (1) In 1831 a professor of Physics at Pisa named Carlo zF ion ion []inside Matteucci used a galvanometer to detect a small electrical current from an injured muscle. Of significant importance, where E is the electromotive force, R is the gas constant, equal though not realized at the time, was that the current moved to 8.3145 V C mol-1 K-1, T is the absolute temperature in from inside the injury to the undamaged muscle surface Kelvin, z is the charge of the ion, and F is Faraday’s constant, outside. This single experiment resurrected the work of 9.6485 x 104 C mol-1 . Galvani and the notion that there was indeed “animal It was shortly after Nernst’s publication that Frederick electricity.” The search to explain it was afoot. George Donnan, a British chemist, began working on the issue of ionic distribution across a membrane and the resulting III. THE GOLDEN AGE OF BIOPHYSICS equilibrium. He argued that a membrane was selectively permeable and that the product of concentrations of ions that With the proof of electrophysiological phenomenon came could diffuse through the membrane is proportional on both the burden of proving the mechanisms responsible for its sides of the membrane. [5] That is existence. In a bold initiative, Hermann von Helmholtz, Émil du Bois-Reymond, and Ernst von Brücke formed an RT []ion RT [ion ] ln 12out = ln out . (2) allegiance and adopted as their decree that "no other forces F []ion −F [ion ] than common physical chemical ones are active within the 12in in organism." [4]. Helmholtz, a prominent German physicist, By absorbing the negative into the logarithm and inverting the directly measured the speed of a nerve pulse at 100 ft/sec, well argument of the logarithm, the equation reduces to within the parameters explainable by electrochemical properties.[4] His collegue du Bois-Reymond had postulated, and later established, that nervous impulses were []ion12in ×[ion ]in =×[]ion1out [ion2]out . (3) electrochemical in nature, and traveled much like an electric pulse through a wire. (The comparison of the nerve pulse This is known as Donnan’s Rule and can be applied to transduction to that of telecommunications could hardly be systems in finite volume equilibrium [5]. If however an anion discounted and it is likely that the two fields were mutually that is not permeable with respect to the membrane is present influential.) He was able to observe a temporary decrease in on one side of the membrane, a cation will diffuse through the the “negative variation”, the injury current described by membrane to maintain electrical neutrality. The presence of Matteucci, though he did not realize the significance of this this impermeable anion results in an unequal distribution of discovery. He further proposed that muscles contained diffusible ions. The result is a chemical gradient of specific positive sources and nerves contained negative sinks. Brücke, 3 ions with respect to the two sides of the membrane. towards the outside. Indeed, this electrical double layer With the identification of the principal ions involved in must also exist in the undamaged fiber, but would become cellular fluids, namely K+, Na+, and Cl-, Julius Bernstein apparent only in response to lesion or stimulation (negative started with the assumption that muscle and nerve fibers are variation). This assumption would imply a theory of enveloped by isolating boundary shells (membranes) that are preexistence; as the semi-permeable membrane plays an permeable only to specific ions. This was the basis of theory essential role in this theory, I will succinctly call it set forth by his childhood friend Ludimar Hermann, who in 'Membrane Theory'.” [6] 1898 proposed his core conductor theory [2]. The theory states that stimulation of the core, which is a conductive core V. THE MEMBRANE surrounded by a non conductive boundary shell, produces an action current. This action current activates adjacent regions In 1925, the first accurate glimpse of the membrane was of the fiber through electrical induction. The theory also offered by Gorter and Grendel. They showed by empirical describes the local excitation in terms of a sudden change or means that the was composed of a alteration, though Bernstein would later prove this assumption [2]. By carefully extracting the lipid from a cell, which was to be false, stating "One can…with some certainty conclude performed by dissolving the cell in organic solvent, they were that a chemical process cannot at all serve as the direct able to isolate only the lipid portion of the cell. They then energy source for the electrical energy of muscle current." [5] + placed the lipid component in water and observed the lipid Bernstein primarily considered K diffusing in the float on the surface, as expected. However, what they also direction of the concentration gradient out of the cell and into observed was that the small polar end of the lipid associated the extra cellular fluid. As negative anions, in particular with the highly polar water, while the long nonpolar strands phosphates, apparently could not pass through the cellular stuck out of the water. Using two wooden floats, they boundary, Bernstein argued that an electrical potential builds squeezed the edge of the lipid layer together and observed the up across the semi-permeable membrane, which would formation of the lipid bilayer [2]. impede further efflux of . This potential Danielli and Davson expanded this idea to include the corresponded exactly to the "resting current". Furthermore, stabilization of this bilayer by a thin layer of protein Bernstein postulated that an excitation of the fibers would lead molecules on both sides of the membrane [7]. This was to a brief loss of the selective membrane permeability, thereby known as the Davson and Danielli model and served as the eliciting "action currents" and a “negative variation” in the accepted model of the cellular membrane for many years. potential. Thus, the first postulation of an action potential, Using a bimolecular layer of lipid 50 angstrom thick with a constant of 5, the of the membrane and the ability of a cell to elicit an electrical response, was -2 formed. would be about 1µF cm , since capacitance is given by kAε C = 0 , where kε is the permittivity of the lipid, A is d 0 area of the membrane in cm2 , and d is the distance between the layers, approximately 50Å [7]. To develop a full picture of the current carried by each of the principal ions across the cellular membrane, the forces acting on each ion and the permeability and conductance of the membrane to that ion must be elucidated. First, the flux due to electric forces is defined by dV Figure 2. Action potential recording from RPD-1, Lymnaea stagnalis. J=−Cµz (4) EMF dx Bernstein would write in 1902 where C is the concentration in moles per cm3, µ is the “A second assumption [in addition to the validity of the dv Nernst equations] concerning the composition of the mobility of the ion, z is the charge of the ion, and concentration chain in muscle is that the electrical potential dx of the lesioned muscle is caused by the electrolytes, in represents the change in over the change in distance. particular by inorganic salts such as K2HPO4, already There is also a flux due to the chemical forces given by contained in the undamaged muscle fiber. Let us imagine that these electrolytes diffuse unhindered from the axial RTdC cross section of the fibrils into the surrounding fluid, while Jchem =− µ . (5) they are prevented from diffusing through the longitudinal Fdx section by an intact sarcoplasmalemma which is impermeable to one kind of ion such as the anion (PO4 - It is now possible to define the total current across the etc.) to a greater or lesser degree. Then an electrical membrane as the superposition of the two fluxes times double layer would emerge at the surface of the fibril, with Faraday’s constant. That is negative charges towards the inside and positive charges 4

equivalent membrane circuit. These two elements may be JJtotal =+EMF Jchem just different aspects of the same membrane mechanism. [9] dC dV (6,7) IJ==F−RTµµ−CFz total total dx dx This is known as the Nernst-Planck flux equation [5]. The term CµFis incremental conductance at one point on the membrane. The conductance of the ion over the whole membrane is then just the integration of the sum of each conductance, remembering that conductance in series adds like resistors in parallel.

VI. ONE SMALL STEP FOR NEUROSCIENCE The has several major parts: the , or body, the , and the . Further divisions in the axon include the , the beginning of the axon core, just after the soma, and the presynaptic knob, a bulb like structure at the end of the axon. An action potential is received by a neuron Figure 3. (A) Directional current flow according to Hermann. (B) Hermann’s cable core model for the from the dendritic region then processed by the soma before axon. (C) Time varying model proposed by Curtis and Cole. [2] passing some measure of transmission “down” the axon. The In the classical image, taken of the front of the axon is typically coated with a sheath that acts as an insulator for the conductive axon. There are, however, gaps, oscilloscope, Curtis and Cole illustrated the first direct called Nodes of Ranvier, which are channel rich and relationship between an increase in conductance (ion serve as transmission enhancers. The gap between the axon of permeability) as a result of increased membrane potential. one neuron and the dendrite of another is called the . (figure 4) However, this created more questions than it This, of course, is common knowledge now, but in the late answered. If the membrane became permeable to all ions, 1920s, this was only beginning to come into focus. why would the E.M.F of the membrane shoot past 0 mV? Advancements in measurement devices by the early 1930s This question would lead to the landmark work of Alan made it possible to make direct measurements from the Hodgkin, , and , and the giant axon, the “conductive cable” portion of the neuron. publication of a series of papers that is perhaps the most Two researchers, Howard J. Curtis and Kenneth S. Cole, celebrated experimentation for the understanding of the ionic revisited Hermann’s to help explain their mechanisms responsible for action potential (AP) propagation. measurements of the membrane impedance. Hermann had suggested that excited regions of an axon adjacent to unexcited regions would generate local circuit currents. He then used the concepts of cable theory to describe the axon as a “leaky” version of a telegraph cable. The Hermann cable representation of an axon consisted of a network of resistors and capacitors in parallel, sandwiched between two resistive rails, representative of the membrane bilayer [2]. Cole and Curtis modeled their membrane with time varying elements to demonstrate that although the capacitance stays constant over time, the conductance and electromotive force vary. (See figure 3) The time varying component of conductance that Curtis and Cole introduced proved to be extraordinarily significant. They concluded that the increase of conductance begins only after a sharp increase in the membrane potential and that this exponentially rising phase of the membrane potential is the Figure 4 Curtis and Cole’s classic picture of the action potential (dotted) and the corresponding rise in discharging of the local circuits. They would go on to suggest membrane conductance.[2] that membrane itself contributed to the net influx of current, HEN THE QUID ANDED ON LYMOUTH OCK though they stopped short of offering an explanation as to VII. W S L P R which ions might be involved in the process. This is John Zachary Young was one of the most important summarized in their 1938 writings as follows: biologists of the twentieth century. He was responsible for discovering a bundle of nerve cell bodies in the squid Loligo For these reasons, we shall assume that the membrane that fused together to form a cable-like fiber core [10]. This resistance and E.M.F. are so intimately related that they core was the and its identification at the should be considered as series elements in the hypothetical Marine Biological Association in Plymouth, UK, spawned a 5 research boom that would lead to the complete quantitative point and approaches zero as the curve approaches a description of the ionic mechanisms of the action potential. maximum or minimum. Hodgkin and Katz identified this Young introduced his work to researchers at the Marine inflexion point as the point where the membrane becomes Biological Laboratory in Woods Hole, MA, in 1936. Among permeable to sodium. those researchers were Curtis and Cole. But it was the work of Hogkin, Huxley, and Katz, at the University of Cambridge Hodgkin and Katz were later joined by A.L. Huxley and that would elevate the squid to its place as the most important together they investigated the current-voltage relationship in neuroscience history. with a device called a , developed by Cole and When was still an undergraduate at the Marmont in 1949 (figure 5) [2]. Modifying the equation for University of Cambridge, he was busy conducting current given earlier to include the ionic contribution of experiments to prove Hermann’s local circuit theory. He took current, it can be shown that an axon and laid it on a cold bar, then stimulated it to form an action potential upstream of the cold bar. (The term upstream dV is used here to denote the fact that action potentials travel in I =Cm+Iion (8) one direction because the refractory period of an AP prevents dt sodium channels from reopening in response to a change in the membrane potential. This will be covered in more detail where Iion is the current due to the ion flow and Cm is the later). Because the membrane was chilled at one point, the dV “voltage-gated” Na+ channels didn’t open there; the membrane capacitance. When is held at 0, that is, the membrane could not change its conductance properties and dt the action potential ceased. But Hodgkin found that even voltage is a constant, or clamped, the current measured is though there was no AP at the cold region, there was one simply the current due to the ion flow. (It is convention to further downstream, beyond the cold bar. He concluded that represent outward current flows as positive, or upward enough current flowed inside the axon from the point before deflection, and inward current flows as negative, or downward the cold block to the point after cooled region, that it brought deflection.) the membrane potential above threshold post cold block, and induced a new AP there [11]. This, however still did not account for the overshoot observed in the action potential beyond the membrane potential Hodgkin, now working with Bernard Katz, proposed a theory for this overshoot. They hypothesized that a large increase in conductance to sodium and influx down its concentration gradient was responsible for the overshoot. Since the squid giant axon could be rolled out like a tube of toothpaste, Hodgkin and Katz did just that very thing, Figure 5. A voltage clamp measures the membrane voltage then passes current back into the membrane squeezing the and blood from the axon. They to maintain the selected signal voltage.[5] measured the concentrations of K+ and Na+ and used these concentrations to calculate equilibrium potentials using the By manipulating Ohm’s Law, V=IR, where V is voltage, I Nernst equation. They found that the resulting potential from is current, and R is resistance, current can be calculated as the the K+ concentration was -75mV while the potential from the potential divided by the resistance. It is equally convenient to Na+ was 55mV [12]. The sodium theory suggested that define a variable g, which is conductance and is given as the potassium was responsible for the but during inverse of resistance. Now Ohm’s law can be rewritten as the action potential, the membrane became more permeable to I=gV. Since conductance is a representation of permeability, sodium, and the maximum potential would tend to the sodium an increase in permeability is seen as an increase in current, equilibrium potential. with the converse also being true. This leads to the set of In an elegant series of experiments Hodgkin and Katz equations offered by Hodgkin, Huxley, and Katz showed that perfusion of the axon with sodium deficient solutions in artificial seawater produced action potentials of I =−gV()V,I =g(V−V),I=g(V−V) (9) marked attenuation while sodium rich solutions in artificial K K K Na Na Na Cl Cl Cl seawater produced increased amplitude in the action potential [12]. What they were also able to show is that the rate of where the subscript identifies the individual ion component, g change in potential with respect to time was also larger for dV is conductance, V is the equilibrium potential, and V subscript sodium rich solutions. Given the relationship IC= , dt is the equilibrium potential of the specified ion [13]. where I is the current and C is the membrane capacitance, the direction of ion flow, i.e., inward or outward, can be determined by the algebraic sign of the derivative. The change in sign of a derivative of a curve is due to an inflexion 6

Figure 6. Current response to voltage clamping at varying potentials. [2]

CTIVE RANSPORT Figure 7. Increased sodium conductance provides initial phase of the action potential, and VIII. A T potassium restores the resting potential.[2] From the mid 1940s through the 1950s, considerable IX. ‘FAT ’, MEPPS, AND PROBABILITY attention was directed toward the cell membrane and the transport mechanisms responsible for the influx of sodium. With the identification of most of the mechanisms Sodium was being ushered into the cell, moving up the responsible for action potentials, Bernard Katz teamed up with instead of down. The idea was put colleague Paul Fatt to investigate curious fluctuations in the forth by WL Dean in 1941 in what he termed the ‘sodium membrane potential of a frog muscle at rest. They had the pump’, but now the search was on to identify how. same shape as an end plate potential, or post synaptic Hans Ussing and K. Zerhan had shown in 1950, using the 24 potential, but were much smaller in amplitude, on the order of radioactive sodium isotope Na , that sodium was being 0.5mV [15]. Even more curious was the fact that these mini actively transported across the membrane. The experiment, end plate potentials, or MEPPs, were caused by the which measured the flux, or transport rate, of radioactive spontaneous release of from the sodium across frog skin, proved that the current model of ending. determining the potential difference created by the flux was in It was well known that end plate potentials were the result need of a correcting factor. They wrote of the release of acetylcholine. (This release is triggered by an action potential arrival at the synapse, the region between It can be demonstrated …that for a free ion species…the nerve cells. Regions are defined with respect to the direction following equation is valid… of propagation of an action potential. If discussion is of the zF Macf−Ψ()1−Ψ2 side before the synapse, that region is described as ‘pre in =o =××o o e RT , where M means Macf in synaptic’; otherwise it is ‘post synaptic.’) Del Castillo and out i i i Katz proposed a quantal release theory, as they suspected

influx and M out means outflux…If the solutions on both acetylcholine was being transmitted across the synapse in sides of the membrane are identical discrete bundles, also known as quanta [16]. The central zF theme to this theory was the idea that the release, and arrival, M −Ψ()1−Ψ2 M in = e RT , in other words: in should be a of one packet of acetylcholine (ACh) was responsible for one MEPP and that multiple packets of ACh were responsible for M out M out function of the potential difference only. It is obvious that an end plate potential (EPP). this equation does not hold for an ion species which is In probability theory, the definition of a single packet with subject to active transport [14]. an outcome of success or failure, and nothing in between, is often given by a Poisson distribution. That is There was indeed evidence of a “channel” for ions to pass x −λ through. Hodgkin and Huxley expanded these concepts and λ e fx()==P(X x)= (10) uncovered the sodium and potassium channels. But it was not x! just a channel; it actively transported sodium across the membrane. The principal components for the formation of the which reads: the probability that the random variable X is action potential were unfolding. exactly equal to some arbitrary value x is the arrival rate λ, or ratio of responses to failures, raised to the x, times the inverse 7 of the exponential of λ, divided by x factorial [17]. (The quantum packet, as well as a host of other important distribution function stems from the law of large numbers and membrane surface features, including ion channels. is found extensively throughout nature in situations where large numbers of elements, such as ions, are interacting.) XI. EXCITATION, INHIBITION, AND THE SYNAPSE This was a statistical problem concerning the ratio of the number of quanta being released from the pre-synaptic side of need inhibition. Consider the movement of a the synapse and the number of arrivals at the post-synaptic sidewinder snake as is scurries across a desert dune. In side. Mathematically, this was a very straightforward addition to contracting, or exciting, the muscles on one side of problem. The question of interest was how were the its body in one particular region, it must inhibit the molecules of ACh being bundled and released in the first contraction of the muscles on the other side of that region place? And for that matter, how were they being absorbed? (figure 9). This allows the locomotion to proceed; if one side is not relaxed the other cannot contract. This is the function X. MEMBRANE MORPHOLOGY of EPSPs, excitatory post-synaptic potentials, and IPSPs, inhibitory post synaptic potentials, and their control is Since the days of recess, children in cold winter climates mediated by several factors. have been daring each other to stick their tongues to metal posts in the dead of winter. The amusement was to watch the struggle of an unwitting child, as he tries desperately to free his subsequently frozen tongue from the pole. What does this have to do with cellular biology? JL Heuser would say, “Plenty!” In 1979, John Heuser developed a technique, similar to the scenario just described, for separating the lipid bilayer of a cell. By cooling a copper plate to -269°C using liquid helium, he created a “frozen pole” for the cell to stick to (figure 8). He first brought the cell, attached to a similarly cold block, in contact with the copper plate [2]. Then he pulled the block away from the plate, exposing the inside (middle) of the lipid bilayer, not unlike separating a crème filled cookie. Figure 9. Network of afferent and efferent pathways and inhibitory interneuron to allow opposed muscle groups to work together. [5]

A central component of the mediation of EPSPs and IPSPs is the affect the membrane potential has on them. It is therefore worthwhile to examine the concepts of .

Time series recording from RPD-1Lymnaea stagnalis 0.02

0

-0.02 ) s t l o -0.04 ial (V t

n -0.06 e t o P -0.08

-0.1

-0.12 0 10 20 30 40 50 60 Time (sec)

Figure 10. Recording from L. stagnalis RPD-1 showing EPSPs. (Small peaks between large peaks)

Hodgkin showed in his experiment using the cold block that small ion fluxes could trigger an action potential. This is

Figure 8. Freeze-fractured image of the separated lipid bilayer. (a) 3 ms before stimulation (b) 5 significant in that even at zero measurable current, minute ion ms after simulation. The larger openings in b are openings into synaptic vesicles. [5] flux is taking place. The reversal potential (RP) is therefore voltage at which no current flows, even though the channels The view was dramatic. Heuser had succeeded in exposing on the cell surface are open. The RP depends on the the surface of a cell from the inside out. Structures were concentrations and relative permeability of all the ions revealed that had been speculated for years, but now there was involved in generating a current. proof of their existence. Working with TS Reese and later with TM Miller, he exposed the formation of the vesicle, the 8

Adapting the Nernst equation to accommodate two It has been shown that can initiate either different types of ions, i.e. sodium and potassium, excitatory or inhibitory postsynaptic potentials. Fatt and Katz discovered that these neurotransmitters were packaged into packets, or quanta. Freeze fracturing and electron microscopy RT PNNa[]aout Ereversal = ln (11) identified these packets as vesicles formed by the cell (see zF PKi[]K n figure 7) The mechanism that determined why and how these vesicles were able to be attached and subsequently released, or where R, T, F, and z have their usual values and significance. alternatively, reabsorbed and recycled, was still unclear. It is straight forward to see that in the simplified model where An action potential induces in presynaptic only one type (same z number) of ion is present, that when the vesicles with the aid of synaptosomal-associated proteins ratio is less than one, the reversal potential is negative [2]. (SNAPs). This is achieved in four steps: 1) the vesicle moves Thus, the post synaptic membrane is able to identify whether into a region on the presynaptic membrane called the active the AP is excitatory, or depolarizing (EPSP) or inhibitory, zone 2) several proteins assist in attaching the vesicle to the hyperpolarizing (IPSP). (The IPSP can be depolarizing if the 3) a complex of SNARE (SNAp REceptor) reversal potential for the channel being opened is more proteins attach, or dock, the vesicle to the membrane and 4) negative than the threshold.) The shape of an IPSP is very rising concentrations in the cell mediates fast fusion similar to an EPSP. of the vesicle to the membrane and the is JS Coombs showed in 1957 that for an EPSP, the response released (figure 11) [18]. is proportional to the stimulus intensity. This is known as spatial summation. Whereas if two EPSPs, either of which is too small in intensity to trigger an action potential on their own, are successive in a short time span, the sum of the two can be large enough to trigger an AP. This is known as temporal summation. This is a key mechanism in the decision making process in the post synaptic neuron, since the arrival of a sufficiently large IPSP, within a short enough time span (less than 2 ms) can cancel the effect of an EPSP, i.e. the summation of a depolarized waveform with a hyperpolarized waveform of equal magnitude is zero [5]. The effect of this situation on an end plate potential, EPP, at a of a sufficiently large IPSP is to relax the muscle. In this example, the motor neuron is the decision maker, much like a logic gate decides based on the Boolean ‘and’ or ‘or’. Figure 11. Artist rendition of SNARE complex “docking” the vesicle and the importance of calcium ion The synapse itself, however, has a morphology that to the process. [19] predisposes the type of response, either excitatory or SNAREs can be categorized as either t-SNAREs, target inhibitory. Using electron microscopy, Gray, and later SNAREs, or v-SNAREs, vesicle SNAREs. Still a further Uchizono, observed shape and thickness differences in known classification names a VAMP, or vesicle associated membrane types of synaptic membranes and clefts. Uchizono went on to protein, which is a type of v-SNARE. The SNARE complex observe the vesicle itself, the packet containing of interest is the synaptobrevin (VAMP), syntaxin (t-SNARE), neurotransmitter, had a distinctive shape, indicative of the and SNAP-25(t-SNARE) complex. [18] type of response they would elicit. Inhibition could be induced at the pre-synaptic region, and was subsequently XIII. DISCUSSION called presynaptic inhibition. Still further designations ensued: Type I and Type II , fast and slow synaptic potentials. There were even Neuroscience is more than a science, it is a quest. It is at different physical mechanisms for intercellular the of human kinds most basic of questions; what is communications. Gap junctions were discovered by Ravel consciousness? The history of the science is fraught with and Karnovsky and indicated that cells could communicate exceptionally talented and adventurous researchers, whose without neurotransmitters. This is termed electrically collective efforts have labored to produce many of the transmitting synapses, or simply electrically coupled synapses. answers we take for granted today. The majority of These gap junctions are much larger than a typical researchers presented in this paper are Nobel laureates, as and can accommodate much large ions. This is what enables their contributions to mankind are of the highest honor. There fast electrical responses that can not be achieved with are, however, a great many scientists whose names are not conventional chemical junctions. listed among the giants of the field not because they did not deserve, but simply because history is kept by those who XII. IT’S A SNAP! (OR A SNARE?) report a version of events. Sir Alan Hodgkin remarked in 1977 that "...the introduction of the squid giant nerve fibre by

J. Z. Young in 1936 did more for neurophysiology and axonology than any other single advance during the past 40 9 years" yet often Young is overlooked in the broad strokes of [17] Leon-Garcia, Alberto. Probability and Random Processes for the the history of neurophysiology. Electrical Engineer. Addison-Wesley Publishing, 1994. It is difficult to imagine where our understanding of [18] Keller, JE and Neale, EA. 2001. The role of the synaptic protein snap- thought and behavior would be were it not for the insight of so 25 in the potency of botulinum type A. J Biol Chem. Apr 20; many great scientists. The action potential, the most basic 276(16):13476-82. [19] http://pharyngula.org/~pzmyers/neuro/synapse/snare.jpg form of cellular communication, is a simple, elegant waveform, yet the production and propagation of the AP is one of the most comprehensive processes in all of animal life. It transcends the boundary, to a certain degree, between and invertebrate and provides more than just a subtle hint that long ago evolution was not quite as diverse as it is today. By citation of previous works, a history is established that lends credence to the methodologies presented as well as the conclusions that have been offered. It is therefore demonstrated that the research, when taken as a whole, provides a verifiably accurate model of the biological and physiological processes with regard to the electrochemical formation and transmission of communicatory signals. We now look at sodium, potassium and calcium channels under high powered electron microscopes and reveal a beautifully complicated network of proteins. And the sodium- potassium-ATPase pump, the existence of which was known long before it was seen, continues to intrigue researchers with complexities we are just now beginning to understand. It is exciting to think what the next hundred years will bring.

REFERENCES

[1] http://www.med.virginia.edu/hs-library/historical/antiqua/galen.htm [2] Hille, Bertil. Ion Channels of Excitable Membranes, 3rd Edition. Sinauer Associates, Inc. Sunderland, MA, 2001. [3] Halliday, David and Resnick, Robert. Fundamentals of Physics, 2nd Edition. John Wiley and Sons, 1981. [4] Watson, Sr., R.I. The great psychologists, 4th edition. New York: J.B. Lippincott Co. 1978. [5] Aidley, David J. The Physiology of Excitable Cells, 3rd Edition. Cambridge University Press, Cambridge, UK, 1989. [6] Bernstein, J. Untersuchungen zur Thermodynamik der bioelektrischen Ströme (1902). Pflügers Archiv 92: 521-562. (Translated) [7] Danielli, J. and Davson, H. A contribution to the theory of permeability of thin films. J. Cell Comp. Physiol 1935; 5: 495-508. [8] Aidley, David J. The Physiology of Excitable Cells, 3rd Edition. Cambridge University Press, Cambridge, UK, 1989. [9] Curtis, Howard J and Cole, Kenneth S. Transverse Electric Impedance of the Squid Giant Axon. J. General Physiol 1938; Vol 21, 757-765. [10] Young, J. Z. 1936. The functioning of the giant nerve fibres of the squid. J. Exp. Biol. 15: 170–185. [11] Hodgkin, A.L. 1937. J. Physiol. London, 90:183,211. [12] Hodgkin, A. L., and B. Katz. 1949. The effect of temperature on the electrical activity of the giant axon of the squid. J. Physiol. 109: 240–249. [13] Hodgkin, A. L., A. F. Huxley, and B. Katz. 1952. Measurement of current-voltage relations in the membrane of the giant axon of Loligo. J. Physiol. 116: 424–448. [14] Ussing, Hans H and Zerhan, K. 1951 Active Transport of Sodium as the Source of Electric Current in the Short-circuited Isolated Frog Skin. Acta Phys. Scand. 23:110-127 [15] Fatt, P and Katz, B. 1952. Spontaneous Subthreshold Activity at Motor Nerve Endings. J. Physiol. 117: 109–128 [16] del Castillo, J and Katz, B. 1954. Quantal Components of the End-Plate Potential. J. Physiol. 124: 560–573.